1. Field of the Invention
This invention relates to a method for measuring cholesterol in low density lipoproteins (hereinafter abbreviated as "ULDL") present in samples derived from living bodies, such as serum, plasma, etc., and a reagent, a reagent composition and a kit which are used for practicing said method.
2. Description of the Related Art
Major components of lipids in serum are cholesterol, triglycerides, phospholipids, etc. These serum lipids bind to apoproteins to form lipoproteins which circulate in the blood. The lipoproteins can be classified by differences in density into high density lipoproteins (HDL), low density lipoproteins (LDL), very low density lipoproteins (VLDL), chylomicrons (CM), etc. Among these lipoproteins, HDL has a function of carrying excess cholesterol deposited on tissues to a liver, and has an anti-arteriosclerotic action. On the other hand, LDL is a major carrier of cholesterol from a liver to each tissue. An increase of LDL seems to have an intimate relation to generation of arteriosclerosis.
Therefore, the cholesterol in LDL (hereinafter abbreviated as "LDL-cholesterol") is regarded as a risk factor for arteriosclerosis and ischemic heart disease (coronary arteriosclerotic disease). Thus, the content of LDL-cholesterol is an important indicator of diagnosis, therapy and prophylaxis of these diseases.
As methods for measuring LDL-cholesterol, there have been known a precipitation method, an ultracentrifugal method, an electrophoresis method, and a Friedewald method. Among these methods, the precipitation method, the ultracentrifugal method and the electrophoresis method have complicated procedures due to pretreatment steps such as separation of LDL from unnecessary lipoproteins other than LDL by precipitation/ultracentrifugation treatments, ultracentrifugation treatment or electrophoresis treatment. Thus, these methods are disadvantageous in that direct measurement using only an autoanalyzer which is widely used in the field of clinical tests is impossible.
On the other hand, according to the Friedewald method known by the Friedewald equation, wherein a total cholesterol value, a HDL-cholesterol value and a triglyceride value are used for computation, it has a problem in that measurement of an accurate LDL-cholesterol amount is impossible in the case of using a sample containing 400 mg/dl or more of triglycerides.
In order to solve the above-mentioned problems, various methods have been developed in recent years. One of them is, for example, the method disclosed in JP-A 7-280812.
This method comprises agglutinating LDL using a agglutinant and/or an antibody, eliminating (consuming) cholesterol contained in lipoproteins other than LDL by introducing it into another reaction system not pertaining to the quantitative reaction, dissolving the agglutinated LDL to such a degree that the quantitative reaction can be carried out, by use of a surfactant and/or an inorganic salt, and measuring the absorbance of the solution by subjecting the LDL-cholesterol to the quantitative reaction.
But since this method employs a three-reagent system or a four-reagent system at the measurement, it can only be applied to a few autoanalyzers which permit employment of such multi-reagent systems. Many autoanalyzers usually used for clinical tests cannot be used for carrying out the measurement by said method because these autoanalyzers can be used for a one-reagent method or a two-reagent method. Further, said method is disadvantageous also in that since a number of reagents are used, reproducibility of measured values is lowered.
As a method for measuring LDL-cholesterol without a troublesome pretreatment, there is the method disclosed in JP-A 58-165800. This method, however, cannot be a practical measuring method for the following reasons: since the using concentrations of, for example, a surfactant and cholesterol esterase (hereinafter abbreviated as "CHE") in reagents are limited, the preparation of the reagents is troublesome; measuring conditions such as pH at the time of measurement, intervals of measuring times, etc. should be strictly set; and since the cholesterol in HDL reacts to some extent, the LDL-cholesterol can be measured only by a kinetic measurement, i.e., a rate assay.